Mitogen-activated protein kinases (MAPKs) are generally expressed in all cell types yet regulate very specific biological responses that differ from cell type to cell type. MAPKs are actually members of a three kinase signaling module composed of the MAPK, MAPK kinase (MKK) and MAPK kinase kinase (MKKK). MKKKs phosphorylate and activate MKKs, which in turn phosphorylate and activate MAPKs. It is the diversity of MKKKs and their different protein-protein interactions and covalent modifications that allows the integration of specific MAPK pathways in the cellular response to diverse stimuli including cytokines, growth factors, antigens, toxins, pharmacological drugs, stress insults, and changes in extracellular matrix and cell- cell interactions. MAPK kinase kinases (MKKKs) are a largely untapped target class for small molecule inhibition to modulate signaling networks, gene expression and potentially human disease. Therefore, the specific aims of this proposal are: 1. Develop robust and reproducible time resolved fluorescence energy transfer (TR-FRET) assays suitable for high-throughput screens (HTS) to identify specific inhibitors for five MAPK kinase kinases. These kinases include MEKK1, MEKK2, MEKK3, MEKK4 and ASK1. These five MAPK kinase kinases are strongly implicated in contributing to tissue remodeling associated with chronic inflammation, pathological hypertrophy, angiogenesis and oxidative stress-induced cell death. No inhibitors for these MAPK kinase kinases have been described resulting in an unmet need for small molecules to explore their cellular and physiological function. 2. Small molecules isolated from HTS or identified from a small directed kinase inhibitor library as inhibitors for a specific MKKK, that is, MEKK1, MEKK2, MEKK3, MEKK4 or ASK1, will be screened against the remaining 14 MAPK kinase kinases, and representative MARK kinases and MAPKs for initial selectivity profiling. 3. Functional cell based screens will be performed for small molecule inhibitors identified for each of the five MAPK kinase kinases (MEKK1, MEKK2, MEKK3, MEKK4 or ASK1). Gene knockouts and RNAi of these MAPK kinase kinases gives a unique phenotype and altered response to specific stimuli providing ready comparison of small molecules to genetic ablation of the kinase for mechanism based selectivity profiling.